The long-term goals of this project are: (a) To understand the structural bases of agonist and antagonist binding and I specificity in the dopamine D2-Iike receptors and related biogenic amine receptors, and (b) To determine how agonist binding is transduced into G-protein activation. During the previous grant period we mapped the entire binding-site crevice of the dopamine D2 receptor, which is lined by the transmembrane segments (This). Our identification of all the residues that are water-accessible in this binding-site crevice led to our identification of structural determinants of pharmacological specificity in the clopamine D2 and D4 receptors. In a D2 receptor background, mutation of a cluster of residues in TM2, TM3, and TM7 to the aligned D4-receptor residues increased the affinity of the mutant D2 receptor for D4-selective ligands by three-orders of magnitude. We studied transduction in the 62 adrenergic receptor where we showed that conformational changes in TM6 are associated with receptor activation and demonstrated that the presence of an "ionic lock" between the cytoplasmic ends of TM3 and TM6 stabilizes the inactive state of the 62 adrenergic receptor. The structural implications of our work are remarkably consistent with the recent high-resolution structure of rhodopsin. Th4 is an exception in that residues facing lipid in rhodopsin are water-accessible in the D2 receptor, and our recent cross-linking data indicate that TM4 may form a D2 receptor homodimer interface. A number of class C GPCRS, such as the GABA8 receptors, are dimers, and there is increasing evidence, both in heterologous expression systems and in native tissue, for homo- and hetero-dimerization of class A receptors, including the dopamine receptors. We hypothesize that ligand binding and receptor I activation is associated with conformational changes at the TM4-dimer interface that are a link in the interaction between the two binding sites. We also hypothesize that just beyond the dimer interface the extracellular loop connecting TM4 and TM5 dives down into the binding-site crevice in the D2-like receptors, as it does in rhodopsin, and contributes to the binding site and to pharmacological specificity. We propose the following specific aims: 1) To map the interaction surface between D2 receptors in the membrane environment by cross-linking endogenous and substituted cysteines.2) To determine whether the heterodimer interface between D2 and D3 receptors as well as the homodimer interface of the homologous 62 adrenergic receptor is similar to the D2 homodimer interface. 3) To assess the effects of ligand-binding and receptor activation on cross-linking as well as the effect of cross-linking on binding and activation. 4) To determine whether interconverting the extracellular loop between Th4 and TM5 and selected amino acid residues therein alters the pharmacological specificity of D2, D3 and D4 dopamine receptors for selective ligands.